I propose to investigate the early proteins encoded by adenovirus type 2, and determine the mechanism by which expression of these proteins effect cellular transformation, viral DNA replication, and RNA processing. One approach is based on a strict genetic analysis. Methods will be developed to introduce point mutations in vitro into early region 1 (the coding region responsible for transformation) and region 4 of the adenovirus genome. This mutated virus will be grown in 293 cells, a human cell line which contains and expresses the left 12% of the viral genome, and in HeLa cells to allow for the selection of host-range and ts mutants. Screening methods for the selection of mutants defective in early region 4 of the viral DNA will be developed. The mutants will be characterized by complementation and marker rescue studies. Ultimately the mutants will be used in dissecting early viral functions and in identifying proteins involved in replication and transcription of viral DNA, and cellular transformation. A complementary approach to the genetic analysis of early viral proteins involves methods that will be developed whereby the cDNA of early viral mRNA, cloned into pBR322, are fused to fragments containing bacterial promoters. The construction of such a plasmid will allow the synthesis of large quantities of viral protein selected for study. In this way the enzymatic activites of these proteins will be correlated with their functions as determined above. In additional experiments, I will locate (by in vitro translation) additional early gene sequences in the viral chromosome by identifying the corresponding early viral proteins encoded in those regions. Thus, these experiments will serve to broaden our understanding of mechanisms employed by the virus to exploit its host, as well as induce cellular transformation. In addition, these results should enhance the value of the adenovirus as a model system in terms of understanding gene expression in eucaryotic cells.